Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
This invention relates to antiretroviral drug susceptibility and resistance tests to be used in identifying effective drug regimens for the treatment of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS). The invention further relates to the means and methods of monitoring the clinical progression of HIV infection and its response to antiretroviral therapy using phenotypic or genotypic susceptibility assays. The Invention also relates to novel vectors, host cells and compositions for carrying out phenotypic susceptibility tests. The invention further relates to the use of various genotypic methodologies to identify patients whose Infection has become resistant to a particular antiretroviral drug regimen. This invention also relates to the screening of candidate antiretroviral drugs for their capacity to inhabit viruses, selected viral sequences and/or viral proteins. More particularly, this invention relates to the determination of nucleoside reverse transcriptase inhibitor resistance using phenotypic susceptibility tests and/or genotypic tests.
HIV infection is characterized by high rates of viral turnover throughout the disease process, eventually leading to CD4 depletion and disease progression. Wei X, Ghosh S K, Taylor M E, et al. (1995) Nature 343, 117-122 and Ho D D, Naumann A U, Perelson A S, et al. (1995) Nature 373, 123-126. The aim of antiretroviral therapy is to achieve substantial and prolonged suppression of viral replication. Achieving sustained viral control is likely to involve the use of sequential therapies, generally each therapy comprising combinations of three or more antiretroviral drugs. Choice of initial and subsequent therapy should, therefore, be made on a rational basis, with knowledge of resistance and cross-resistance patterns being vital to guiding those decisions. The primary rationale of combination therapy relates to synergistic or additive activity to achieve greater inhibition of viral replication. The tolerability of drug regimens will remain critical, however, as therapy will need to be maintained over many years.
In an untreated patient, some 100 new viral particles are produced per day. Coupled with the failure of HIV reverse transcriptase (RT) to correct transcription errors by exonucleolytic proofreading, this high level of viral turnover results in 104 to 103 mutations per day at each position in the HIV genome. The result is the rapid establishment of extensive genotypic variation. While some template positions or base pair substitutions may be more error prone (Mansky L M, Temin H M (1995) J Virol 69, 5087-5094) (Schinazi R F, Lloyd R M, Ramanathan C S, et al. (1994) Antimicrob Agents Chemoter 38, 268-274), mathematical modeling suggests that, at every possible single point, mutation may occur up to 10,000 times per day in infected individuals.
For antiretroviral drug resistance to occur, the target enzyme must be modified while preserving its function in the presence of the inhibitor. Point mutations leading to an amino acid substitution may result in change in shape, size or charge of the active site, substrate binding site or surrounding regions of the enzyme. Mutants resistant to antiretroviral agents have been detected at low levels before the initiation of therapy. (Mohri H, Singh M K, Ching W T W, et al. (1993) Proc Natl Acad Sci USA 90, 25-29) (Nxc3xa1jera I, Richman D D, Olivares I, et al. (1994) AIDS Res Hum Retroviruses 10, 479-1488) (Nxc3xa1jera I, Holguin A, Quixc3x1ones-Mateu E, et al. (1995) J Virol 69, 23-31). However, these mutant strains represent only a small proportion of the total viral load and may have a replication or competitive disadvantage compared with wild-type virus. (Coffin J M (1995) Science 267, 483-489). The selective pressure of antiretroviral therapy provides these drug-resistant mutants with a competitive advantage and thus they come to represent the dominant quasispecies (Frost S D W, McLean A R (1994) AIDS 8, 323-332) (Kellam P, Boucher C A B, Tijnagal J M G H (1994) J Gen Virol 75, 341-351) ultimately leading to drug resistance and virologic failure in the patient.
Nucleoside Reverse Transcriptase Inhibitors
Seven nucleoside analogue HIV reverse transcriptase Inhibitors (zidovudine (ZVD: Retrovir, Glaxo Wellcome, Uxbridge, UK), zalcitabine (ddC: HIVID, Hoffman-LaRoche, Basle, Switzerland), didanosine (ddI: Videx, Bristol-Myers Squibb, Syracuse, N.Y., USA), stavudine (d4T: Zerit, Bristol-Myers Squibb, Syracuse, N.Y., USA), and lamivudine (3TC, Epivir), abacavir (ABC, Ziagen, Glaxo Wellcome), and adefovir (ADV, Preveon, Gilead Sciences) are currently licensed in Europe and the USA. Additionally, three NNRTIs, nevirapine (Viramune, Boehringer Ingelheim, Ingelheim am Rhein, Germany) and delavirdine (Rescriptor, Pharmacia and Upjohn, Kalamazoo, Mich., USA) and efavirenz (EPV,) are licensed in the USA. All these agents have demonstrated at least short-term antiviral activity and, therefore, it is not surprising that, as they exert a selective pressure on HIV, drug-resistant mutants arise during therapy. Whilst these drugs are normally used in combination regimens, many of the available resistance data arise from phase I/II monotherapy studies. Mutations observed during monotherapy may not accurately reflect mutations responsible for resistance that develops in the presence of pressure from several agents acting at the same site and, hence, on the same gene.
Novel Mutations
Whilst patterns of genotypic mutations associated with changes in phenotypic resistance to the leading reverse transcriptase inhibitors (RTIs) are established from both in-vitro and in-vivo work, other, rarely reported, resistance mutations may arise occasionally during clinical studies. Isolates with a unique pattern of amino acid substitutions at codons 62, 75, 77, 116, and 115 have been identified in patients receiving prolonged combination therapy with ZDV plus ddI or ddC: these isolates are resistant to both drugs and there is cross-resistance to stavudine and partial cross-resistance to 3TC. No consistent genotypic change has been associated with phenotypic d4T resistance or, indeed, loss of virological effect of this compound.
Mutations to Nucleoside Analogue RT Inhibitors Zidovudine
HIV variants with decreased susceptibility to ZDV were first reported in 1989; in some isolates, greater than 100-fold increases in the concentration of ZDV were required to inhibit viral replication by 50% (Larder B A, Darby G, Richman D D (1989) Science 243, 1731-1734). The ZDV-resistant phenotype appears to be reasonably stable in vivo, with resistant virus sometimes being detected up to 1 year after cessation of therapy, (Boucher C A, O""Sullivan E, Mulder J W et al. (1992) J Infect Disease 165, 105-110) and despite treatment with didanosine (Smith M S, Koerber K L, Pagano J S, (1994) J Infect Disease 169, 184-188).
Nucleotide sequencing of HIV RT has revealed a number of mutations which can influence viral sensitivity to ZDV and which may be used as genotypic markers for the presence of ZDV resistance (Kellam P, Boucher C A B, Tijnagal J M G H et al. (1994) J Gen Virol 75, 341-351) (Boucher C A B, Tersmette M, Lange J M A, et al. (1990) Lancet 336, 585-590) (Lopez-Galindez C, Rojas J M, Najera R, et al. (1991) PNAS 88, 4280-4284). A range of mutants with increasing levels of resistance appear in an ordered manner, with the sequential appearance of these mutations being associated with incremental reductions in viral sensitivity to ZDV (id) (Larder B A, Kellam P, Kemp S D, (1991) AIDS 5, 137-144). A substitution at codon 70 (Arg70xe2x86x92Lys) may be transiently dominant and appears critical to virological failure during ZDV monotherapy (DeJong M D, Veenstra J, Stilianakis N I, et al. (1996) PNAS 93, 9501-9506). Continued ZDV therapy selects for a further mutation at codon 215, which appears to be a more stable variant, though both Thr215xe2x86x92Tyr and Thr215xe2x86x92Phe substitutions have been described and may coexist (Mayers D L, McCutchan F E, Sanders-Buell E E, et al. (1992) J Acq Imm Def Synd 5, 749-759). Virus with additional mutations may then appear, most commonly a substitution at codon 41 (Met41xe2x86x92Leu), followed by further additional mutations at codons 67, (Asp67xe2x86x92Asn) and 219 (Lys219xe2x86x92Gln) or the reappearance of the codon 70 mutation
Site-directed mutagenesis techniques have been used to assess the interactions resulting from the different mutations (id). These demonstrated that high-level resistance to ZDV (IC50 greater than 1 xcexcM) is typically associated with the presence of multiple mutations. Although frequently synergic, mutations may also be antagonistic. For example, a mutation at codon 74 (Leu74xe2x86x92Val) observed during therapy with ddI or ddC has been noted to be antagonistic to the ZDV 215 mutation in vitro, reducing the degree of resistance to ZDV (St Clair M H, Martin J L, Tudor-Williams G, et al. (1991) Science 253, 1557-1559). Antagonism of the 215 mutation in vitro has also been reported by the codon 181 mutation selected for by most NNRTIs and the mutation at codon 184 seen with lamivudine and, less frequently, ddC and ddI (Larder B A, (1992) Antimicrob Agents Chemother 36, 2064-2669) (Boucher C A B, Cammack N, Schipper P, et al. (1993) Antimicrob Agents Chemother 37, 2231-2234) (Tisdale M, Kemp S D, Parry N R, et al. (1993) PNAS 90, 5653-5656) (Larder B A, Kemp S D, Harrigan P R (1995) Science 269, 696-699) (Zhang D, Caliendo A M, Eron J J, et al. (1994) Antimicrob Agents Chemother 38, 282-287). However, novel mutation patterns or additional xe2x80x98compensatoryxe2x80x99 mutations may be observed in vivo during combination therapy facilitating dual or multi-drug resistance (see below).
Viral strains resistant to ZDV exhibit cross-resistance to other nucleoside analogues containing the 3xe2x80x2-azido group such as 3xe2x80x2-azido-2xe2x80x2,3xe2x80x2-dideoxyuridine (AZU) (Rooke R, Parniak M A, Tremblay M, et al. (1991) Antimicrob Agents Chermother 35, 988-991). Cross-resistance to stavudine, a thymidine-based analogue which lacks a 3xe2x80x2-azido moiety, has also been reported by one group in both a laboratory strain of HIV and one of 11 clinical isolates (ibid). Most investigators have found no evidence that mutations selected for during ZDV monotherapy influence sensitivity to ddI, ddC or 3TC (Rooke R, Tremblay M, Soudeyns H, et al. (1989) AIDS 3, 411-415) (id) (Larder B A, Chesebro B, Richman D D (1990) Antimicrob Agents Chemother 34, 436-441) (id) (Dimitrov D H, Hollinger F B, Baker C J, et al. (1993) J Infect Disease 167, 818-823). However, resistance to ddI has been rarely reported after prolonged ZDV therapy (id) (Japour A J, Chatis P A, Eigenrauch H A, et al. (1991) PNAS 88, 3092-3096), and one report has suggested that, for each ten-fold decrease in ZDV sensitivity in clinical isolates, there is a corresponding 2.2-fold reduction in susceptibility to ddI and two-fold decrease in sensitivity to ddC (Mayers D L, Japour A J, Arduino J M, et al. (1994) Antimicrob Agents Chemother 38, 307-314). Furthermore, patients with ZDV resistance at baseline are significantly less likely to achieve an RNA response after the addition of ddC or ddI than those with wild-type virus at baseline (Holodniy M, Katzenstein D, Mole L, et al. (1996) J Infect Disease 174, 854-857).
Zalcitabine And Didanosine
Resistance to ddI is mediated through a Leu74xe2x86x92Val mutation which produces a six-fold to 26-fold reduction in sensitivity, but may partially restore susceptibility to ZDV in vitro by antagonism of the codon 215 mutation. This mutation also reduces sensitivity to ddC by around ten-fold (id). The frequency of the codon 74 mutation has been reported to have increased from zero at the start of therapy to 56% at week 24 in a group of 64 persons with a mean baseline CD4 cell count of 129/mm who switched to ddI having previously received ZDV (Kozal M J, Kroodsma K, Winters M A, et al. (1994) Annals Intern Med 121, 263-268). Similarly, in a mixed population of both treatment-naive and ZDV-experienced patients with CD4 cell counts of 200-500/mm who received ddI monotherapy in the ACTG 143 study, 17 of 26 isolates had mutations at codon 74 at 1 year. Mutant codon 74 arose in only two of the 55 patients in this study who received ZDV/ddI combination therapy (Shafer R W, Iversen A K N, Winters M A, et al. (1995) J Infect Disease 172, 70-78).
Virus with a mutation at codon 65 (Lys65xe2x86x92Arg) has been isolated from several patients receiving long-term treatment with ddI or ddC. This is associated with a three-fold to five-fold increase in the IC50 of ddI with a five-fold to ten-fold reduction in ddC sensitivity and a 20-fold reduction in susceptibility to 3TC (id) (Gu Z, Gao Q, Fang H, et al. (1994) Antimicrob Agents Chemother 38, 275-281. A mutation at codon 69 (Thr69xe2x86x92Asp), which leads to a five-fold reduction in sensitivity to ddC but does not appear to result In cross-resistance to other nucleoside analogues, is the most frequent mutation selected for by ddC in vivo (id) (Fitzgibbon J E, Howell R M, Haberzettl C A, et al. (1992) Antimicrob Agents Chemother 36, 153-157). The development of resistance to ddC has recently been reviewed elsewhere (Craig C, Moyle G (1997) AIDS 11, 271-279).
Combination Therapyxe2x88x92Zidovudine+Zalcitabine or Didanosine
Combination therapy with ZDV/ddC or ZDV/ddI may influence the rate of emergence of resistance and may suppress some of the mutations observed during monotherapy but may result in the appearance of novel (and hence possibly more compromised) mutational patterns.
Novel mutation patterns may emerge during combination therapy. Isolates with a unique pattern of amino acid substitutions at codons 62, 75, 77, 116, and 115 have occasionally been identified in patients receiving prolonged combination therapy with ZDV plus ddI or alternating ZDV/ddC: these are resistant to both drugs (id) (Shafer R W, Kozal M J, Winters M A, et al. (1994) J Infect Disease 169, 722-729) (Shirasaka T, Kavlick M F, Ueno T, et al. (1995) PNAS 92, 2398-2402) and confer cross-resistance to stavudine and partial cross-resistance to 3TC. The frequency in persons treated for  greater than 1 year with ZDV. ddU ranges from 0 to  greater than 10% (ibid). Mutations selected by 3TC (184Val) and nevirapine (181Cys) may readily be added to this background in vitro (Shafer R W, Winters M A, Iversen A K N, et al. (1996) Antimicrob Agents Chemother 40, 2887-2890) and 184Val and 103Asp (for loviride resistance) being reported in vivo (Schmit J C, Cogniaux J, Hermans P, et al. (1996) J Infect Disease 174, 962-968). While these virus mutations appear to be replication competent in the presence of drug, the likely reason these novel mutations are not seen during monotherapy probably relates to their failure to compete with those mutants that become dominant.
Lamivudine
Resistance to 3TC occurs rapidly in vivo with a substitution at codon 184 (most commonly Met184xe2x86x92Val) (id) (Kuritzkes D R, Quinn J B, Benoit S L, et al. (1996) AIDS 10, 975-981) (Bartlett J A, Benoit S L, Johnson V A, et al. (1996) Annal Intern Med 125, 161-172) (Eron J J, Benoit S L, Jemsek J, et al. (1995) NEJM 333, 1662-1669) (Katlama C, Ingrand D, Loveday C, et al. (1996) JAMA 276, 118-125) (Staszewski S, Loveday C, Picazo J J, et al. (1996) JAMA 276, 111-117) being observed during both monotherapy and combination therapy and its appearance being temporally associated with at least partial virological failure (id) (Moyle G J (1996) Drugs 52, 168-185) (Goulden M G, Cammack N, Hopewell P L, et al. (1996) AIDS 10, 101-102). This mutation leads to high-level resistance to 3TC (500-fold to 1000-fold increase in IC50), as well as some cross-resistance to both ddI and ddC (four-fold to eight-fold reductions in susceptibility) (id) (Gu Z, Gao Q, Li X, et al. (1992) J Virol 66, 7128-7135) In vitro this mutation may antagonize ZDV (id) (id) (id), although dual ZDV/3TC resistance has been reported both in vitro and in clinical isolates (id). Other, possibly compensatory, mutations such as at codon 135 or 333 may be required for dual ZDV/3TC resistance, an issue that is currently under investigation (id). When 3TC was added to patients pre-treated with ZDV in study NUCA3002, phenotypic 3TC resistance developed in 82% of 33 patients by week 12. Of the ten patients with ZDV resistance at baseline (as defined by an IC50 greater than 0.2 mM) who developed 3TC resistance, four had isolates that were more sensitive to ZDV whilst six patients had dual ZDV/3TC resistance, suggesting that viral resensitization to ZDV is not universal in vivo.
Stavudine
In vitro selection of HIV resistant to d4T, confirmed by site-directed mutagenesis, has identified a mutation at codon 75 (Val75xe2x86x92Thr) which confers a seven-fold increase in IC50, as well as reduced susceptibility to both ddI and ddC (Lacey S F, Larder B A (1994) Antimicrob Agent Chemother 38, 1428-1432). A mutation at codon 50 leading to a 30-fold reduction in d4T sensitivity, but which does not appear to confer cross-resistance to other nucleoside analogues has also been observed in vitro (Gu Z, Gao Q, Fank H, et al. (1994) Leukemia 8, Suppl. 1, 166-169). In vivo, however, a range of amino acid changes, including the codon 75 mutation but not the codon 50 substitution, have been reported. The maximum decrease in sensitivity to d4T seen in 13 ZDV-naive patients followed for 18 to 22 months was 12-fold. However, five patients developed nine-fold to 176-fold reductions in ZDV sensitivity and three subjects developed seven-fold to 29-fold decreases in susceptibility to ddI (Lin P F, Samanta H, Rose R E, et al. (1994) J Infect Disease 170, 1157-1164), suggesting use of d4T may limit subsequent therapeutic options in some patients. No consistent mutation pattern for resistance to d4T has, therefore, been established.
Abacavir
In vitro selection of HIV strains resistant to abacavir, confirmed by site-directed mutagenesis, has shown that individual mutations cause only low level resistance to abacavir. Multiple mutations (at least three) are required to produce 10-fold resistance. M184V is the most common resistance mutation selected in vitro in the presence of abacavir and results in a 2-5 fold decrease in susceptibility. Mutations at L74V and F115Y were also shown to contribute to loss of susceptibility to abacavir (Tisdale M, Alnadaf T, Cousens D (1997) Antimicrob Agent Chemother 41, 1094-1098). Cross resistance to ddC and ddI were observed but not to d4T or ZDV. Resistance in HIV derived from patient virus populations has been ascribed to mutations previously associated with NRTI-resistance. A combination of ZDV-resistance mutations (M41L, L210W, T215Y) plus a 3TC-resistance mutation (M184V) showed an eight fold reduction in susceptibility to abacavir. The multi-nucleoside resistance complex (A62V, V75I, F77L, Y116F and Q115M) was associated with a 17 fold reduction in susceptibility (Lanier R, Danehower S, Daluge S, et. al. (1998) 2nd International Workshop on HIV Drug Resistance and Treatment Strategies).
Adefovir
In vitro selection in the presence of adefovir resulted in either a K65R or a K70E mutation appearing which confers 16- or 9-fold reduction in susceptibility to adefovir. Studies in patients have reported the appearance of the K70E mutation but not the K65R mutation. Many AZT-resistant, 3TC-resistant and multi-drug resistant viruses remain sensitive to adefovir (Mulato A S, Lamy P D, Miller M D et. al. (1998) Antimicrob Agent Chemother 42, 1620-1628).
Clinical Significance of Resistance
Choice of initial and subsequent therapy for HIV infection should be uncompromising in terms of activity but also planned and based rationally on knowledge of resistance and cross-resistance patterns to maintain a wide base of future therapy options.
It is an object of this invention to provide a drug susceptibility and resistance test capable of showing whether a viral population in a patient is resistant to a given prescribed drug. Another object of this invention is to provide a test that will enable the physician to substitute one or more drugs in a therapeutic regimen for a patient that has become resistant to a given drug or drugs after a course of therapy. Yet another object of this invention is to provide a test that will enable selection of an effective drug regimen for the treatment of HIV infections and/or AIDS. Yet another object of this invention is to provide the means for identifying the drugs to which a patient has become resistant, in particular identifying resistance to nucleoside reverse transcriptase inhibitors. Still another object of this invention is to provide a test and methods for evaluating the biological effectiveness of candidate drug compounds that act on specific viruses, viral genes and/or viral proteins particularly with respect to viral drug resistance associated with nucleoside reverse transcriptase inhibitors. It is also an object of this invention to provide the means and compositions for evaluating HIV antiretroviral drug resistance and susceptibility. This and other objects of this invention will be apparent from the specification as a whole.
The present invention relates to methods of monitoring, using phenotypic and genotypic methods, the clinical progression of human immunodeficiency virus infection and its response to antiviral therapy. The invention is also based, in part, on the discovery that genetic changes in HIV reverse transcriptase (RT) which confer resistance to antiretroviral therapy may be rapidly determined directly from patient plasma HIV RNA using phenotypic or genotypic methods. The methods utilize nucleic acid amplification based assays, such as polymerase chain reaction. Herein after such nucleic acid amplification based assays will be referred to as PCR based assays. Alternatively, methods evaluating viral nucleic acid or viral protein in the absence of an amplification step could utilize the teaching of this invention to monitor and/or modify antiretroviral therapy. This invention is based in part on the discovery of a mutation/insertion at codon 69 either alone or in combination with a mutation at codon 41 and 215 of HIV reverse transcriptase in nucleoside reverse transcriptase inhibitor (NRTI) treated patient(s) in which the presence of the mutations correlate with a decrease in d4T susceptibility, and a decrease in susceptibility to AZT, ddC, ddI, 3TC, and abacavir. The mutations were found in plasma HIV RNA after a period of time following initiation of therapy. The development of the mutation/insertion at codon 69 in addition to the mutation at codon 41 and 215 in HIV RT was found to be an indicator of the development of resistance and ultimately of immunological decline. More specifically the mutation/insertion at codon 69 in RT (T69SSA, T69SSG, T69SSS) may also be associated with mutations associated with resistance to AZT (e.g. M41L, L210W, T215Y) and 3TC (M184V) or ddI/ddC (L74V) which correlate with a decrease in NRTI-susceptibility, including a decrease in d4T susceptibility and a decrease in susceptibility to AZT, ddC, ddI, 3TC and abacavir. It was also found the mutation/insertion at codon 69 in RT (T69SSA, T69SSG, T69SSS) may be associated with mutations associated with resistance to multi-NRTIs at codon 62 (e.g. A62V) and/or a novel mutation at codon 75 (e.g. V75M). It was observed for the first time that the mutation/insertion at codon 69 (T69SSG) and the mutation at codon 75 (V75M) was associated with decreased susceptibility to d4T (three fold) and substantial decreases in AZT susceptibility (thirty fold). This invention is based in part on the discovery of mutations associated with multi-NRTI resistance at codons 62, 75, 77, 116, and 115 of RT discovered to occur in nucleoside reverse transcriptase inhibitor (NRTI) treated patients in which the presence of the mutation correlates with decreased susceptibility to d4T, ddC, ddI and AZT. It has also been discovered that mutations specifically associated with resistance to: AZT at codons 41, 67, 210, 215 and 219 (e.g. M41L, D67N, L210W, T215Y, K219Q); 3TC at codon 184 (M184V); ddC at codon 69 (T69D); or a novel mutatIon at codon 215 (T215V) may accompany the mutations associated with multi-NRTI resistance at codons 62, 75, 77, 116, or 115 which correlate with decreased susceptibility to d4T, ddC, ddI and AZT. This invention is based in part on the discovery of four or more mutations associated with AZT resistance selected from the group consisting of codons 41, 67, 70, 210, 215 and/or 219 (e.g. M41L, D67N, K70R, L210W, T215Y/F, K219Q) either alone or in combination with a mutation at codon 74 (associated with ddI resistancexe2x80x94V74I), 69 (associated with ddC resistancexe2x80x94T69D), 75 (V75M, V75S) and/or 219 (K219N) of HIV reverse transcriptase in nucleoside reverse transcriptase inhibitor treated patient(s) in which the presence of the mutations correlate with a decrease in d4T susceptibility. The mutations were found in plasma HIV RNA after a period of time following initiation of NRTI therapy. It was observed through the construction, by site directed mutagenesis, of resistance test vectors containing the single site mutation at codon 75 (V75I) did not alter d4T susceptibility but increased AZT susceptibility. It was also observed using site directed mutagenesis that the single site mutation at codon 115 (Q151M) reduced d4T and AZT susceptibility. Yet an additional observation of the present invention was that the double site mutation at codons 75 and 115 (V75I+Q151M) reduced d4T and AZT susceptibility. It was also discovered using site directed mutagenesis that resistance test vectors containing five (M41L, D67N, K70R, T215Y, K219Q) or six (M41L, D67N, K70R, L210W, T215Y, K219Q) AZT resistance associated mutations showed reduced susceptibility to d4T and AZT. It was also observed that resistance test vectors containing single site mutations at codons 62, 69 and 75 (A62V, T69SSA, V75I, V75V) did not reduce d4T susceptibility. However, it was observed that the single site mutation at codon 75 (V75I) or (V75V) increased AZT susceptibility slightly. The T69SSA single site mutation reduced AZT susceptibility slightly while the A62V mutation had no effect on AZT susceptibility. In yet further studies using site directed mutagenesis, it was observed that resistance test vectors containing double site mutations at codons 62 and 69 (e.g. A62V+T69SSA) did not reduce d4T susceptibility more than the T69SSA mutation alone, but further reduce AZT susceptibility due to the T69SSA mutation alone. In still further studies using site directed mutagenesis, it was observed that resistance test vectors containing double site mutations at codons 62 and 75 (e.g. A62V+V75I) had no effect on d4T susceptibilIty. It was also found that the A62V mutation did not alter the reduced AZT susceptibility caused by the V75I mutation. In yet further studies using site directed mutagenesis, it was observed that a combination of three mutations at codons 62, 69 and 75 (e.g. A62V+T69SSA+V75I) did not reduce d4T susceptibility more than the T69SSA mutation alone. It was also observed in the case of the combination of three mutations at codons 62, 69 and 75 (e.g. A62V+T69SSA+V75I) that the V75I mutation completely suppressed the reduced AZT susceptibility caused by the combination of A62V and T69SSA mutations. In still further studies using site directed mutagenesis, it was observed that resistance test vectors containing three mutations at codons 41, 69 and 215 (e.g. M41L+T69SSA+T215Y) showed a significant decrease in both d4T and AZT susceptibility. In yet further studies using site directed mutagenesis, it was observed that a combination of four mutations at codons 41, 62, 69 and 215 (e.g. M41L+A62V+T69SSA+T215Y) did reduce d4T susceptibility more than the T69SSA mutation alone or the T69SSA+A62V double mutant. It was also observed in the case of the combination of four mutations at codons 41, 62, 69 and 215 (e.g. M41L+A62V+T69SSA+T215Y) that the combination of all four mutations reduced AZT susceptibility more than the combination of M41L and T215Y mutations alone. In yet further studies using site directed mutagenesis, it was observed that a combination of five mutations at codons 41, 62, 69, 184 and 215 (e.g. M41L+A62V+T69SSA+M184V+T215Y) did reduce d4T susceptibility more than the T69SSA mutation alone or the A62V+T69SSA double mutant. It was also observed in the case of the combination of five mutations at codons 41, 62, 69, 184 and 215 (e.g. T41L+A62V+T69SSA+M184V+T215Y) that the M184V mutation suppressed the reduced AZT susceptibility caused by the combination of M41L, A62V, T69SSA and T215Y mutations. In yet another study using site directed mutagenesis the T69SSA mutation in a clone of a patient""s virus was reverted (T69SSAxe2x86x92SSA69T). Reversion of the T69SSA mutation reduced d4T resistance (i.e. increased susceptibility) and also reduced AZT resistance (i.e. increased susceptibility).
In yet further studies using site directed mutagenesis, it was observed that the introduction of the L210W mutation with mutations at 41, 69, and 215 (e.g. M41L+T69SSA+T215Y) resulted in a substantial decrease in susceptibility to AZT compared to the 140-fold decrease in susceptibility observed for AZT without the 210 mutation. In still further studies using site directed mutagenesis, it was observed that four mutations at codons 41, 62, 69 and 215 (e.g. M41L+A62V+T69SSA+T215Y) showed a substantial decrease in AZT susceptibility (greater than 1000-fold) and only slight decreases in susceptibility to the other NRTIs. In yet further studies using site directed mutagenesis, it was observed that the introduction of the L210W mutation with four mutations at codons 41, 62, 69 and 215 (e.g. M41L+A62V+T69SSA+T215Y) had little effect on drug susceptibility and showed a resistance profile similar to the profile obtained for when only the four mutations were present. In yet further studies using site directed mutagenesis, it was observed that the introduction of the T215Y mutation with mutations at 62 and 69 (e.g. A62V+T69SSA) resulted in a substantial decrease in susceptibility to AZT (greater than 1000-fold) compared to the 7-fold decrease in susceptibility observed for AZT without the 215 mutation. It was also observed that the introduction of the L74V mutation with mutations at 62 and 69 (e.g. A62V+T69SSA) resulted in a shift back to wild-type susceptibility for AZT. In yet further studies using site directed mutagenesis, it was observed that the introduction of the V75M mutation with four mutations at codons 41, 69, 210 and 215 (e.g. M41L+T69SSA+L210W+T215Y) had little effect on drug susceptibility and showed a resistance profile similar to the profile obtained when only the four mutations were present.
In a further embodiment of the invention, PCR based assays, including phenotypic and genotypic assays, may be used to detect mutations at codon 69 in combination with mutations at other codons including 41 and/or 215 of HIV RT which correlate with a specific pattern of resistance to antiretroviral therapies and subsequent immunologic decline. More specifically in yet another embodiment of the invention PCR based assays, including phenotypic and genotypic assays, may be used to detect mutations at codon 69 (T69SSA, T69SSG, T69SSS) in combination with mutations at other codons including 41 (M41L), 210 (L210W), 215 (T215Y), 184 (M184V) and/or 74 (L74V) of HIV RT which correlate, as described herein, with a specific pattern of resistance to antiretroviral therapies and subsequent immunologic decline. In yet another embodiment of the invention, PCR based assays, including phenotypic and genotypic assays, may be used to detect mutations at codon 62, 75, 77, 116 or 115 either alone or in combination with mutations at other codons including 41, 67, 210, 215, 219, 184, 69 and/or 215 of HIV HT which correlate, as described herein, with resistance to antiretroviral therapies and immunologic decline. Examples of the mutations at the aforementioned codons include, but are not limited to (A62V, V75I, F77L, F116Y, Q151M) and (M41L, D67N, L210W, T215Y, K219Q, M184V/I, T69D, T215Y). In yet another embodiment of the invention, PCR based assays, including phenotypic and genotypic assays, may be used to detect four or more mutations in RT at codons in the group consisting of 41, 67, 70, 210, 215 and/or 219 (e.g. M41L, D67N, K70R, L210W, T215Y/F, K219Q) either alone or in combination with mutation at codon 74 (V74I), 69 (T69D), 75 (V75M, V75S) and/or 219 (K219N) of HIV RT which correlates, as described herein, with resistance to antiretroviral therapy and immunologic decline. Once mutations at codon 69 either alone or in combination with mutation at codon 41 and 215 of HIV RT in a patient undergoing NRTI antiretroviral therapy, an alteration in the therapeutic regimen must be considered. Similarly, once mutations at codon 69 and/or 41, 210, 215, 184 and/or 74 have been detected in a patient undergoing certain NRTI antiretroviral therapy, an alteration in the therapeutic regimen must be considered. Similarly, once mutations at codon 62, 75, 77, 116 and/or 115 either alone or in combination with mutations associated with resistance to AZT, 3TC, ddC or a mutation T215V has been detected in a patient undergoing certain NRTI antiretroviral therapy, an alteration in the therapeutic regimen must be considered. Similarly, once four or more mutations associated with AZT resistance selected from the group consisting of 41, 67, 70, 210, 215 and/or 219 either alone or in combination with a mutation at codon 74 (V741), 69 (T69D), 75 (V75M, V75S) and/or 219(K219N) has been detected in a patient undergoing certain NRTI antiretroviral therapy, an alteration in the therapeutic regimen must be considered. PCR based assays, including phenotypic and genotypic assays, may be used to detect mutations at codon 69 in combination with mutations at other codons including 41 and/or 215 of HIV RT which correlate with a specific pattern of resistance to antiretroviral therapies and subsequent immunologic decline. Similarly, PCR based assays, including phenotypic and genotypic assays, may be used to detect mutations at codon 69 in combination with mutations at other codons including 41, 219, 215, 184 and/or 74 of HIV RT which correlate with a specific pattern of resistance to antiretroviral therapies and subsequent immunologic decline. PCR based assays, including phenotypic and genotypic assays, may be used to detect mutations at codon 69 in combination with mutations at other codons including 62 and/or 75 of HIV RT which correlate with a specific pattern of resistance to antiretroviral therapies and subsequent immunologic decline. PCR based assays, including phenotypic and genotypic assays, may be used to detect mutations at codon 62, 75, 77, 116 and 115 either alone or in combination with mutations at other codons including 41, 67, 210, 215, 219, 184, 69 and/or T215V of HIV RT which correlate with a specific pattern of resistance to antiretroviral therapies and subsequent immunologic decline. The timing at which a modification of the therapeutic regimen should be made, following the assessment of the antiretroviral therapy using PCR based assays, may depend on several factors including the patient""s viral load, CD4 count, and prior treatment history.
In another aspect of the invention there is provided a method for assessing the effectiveness of a nucleoside reverse transcriptase antiretroviral drug comprising: (a) introducing a resistance test vector comprising a patient-derived segment and an indicator gene into a host cell; (b) culturing the host cell from step (a); (c) measuring expression of the indicator gene in a target host cell wherein expression of the indicator gene is dependent upon the patient derived segment; and (d) comparing the expression of the indicator gene from step (c) with the expression of the indicator gene measured when steps (a)-(c) are carried out in the absence of the NRTI anti-HIV drug, wherein a test concentration of the NRTI, anti-HIV drug is presented at steps (a)-(c); at steps (b)-(c); or at step (c).
This invention also provides a method for assessing the effectiveness of non-nucleoside reverse transcriptase antiretroviral therapy in a patient comprising: (a) developing a standard curve of drug susceptibility for an NRTI anti-HIV drug; (b) determining NRTI anti-HIV drug susceptibility in the patient using the susceptibility test described above; and (c) comparing the NRTI anti-HIV drug susceptibility in step (b) with the standard curve determined in step (a), wherein a decrease in NRTI anti-HIV susceptibility indicates development of anti-HIV drug resistance in the patient.
This invention also provides a method for evaluating the biological effectiveness of a candidate HIV antiretroviral drug compound comprising: (a) introducing a resistance test vector comprising a patient-derived segment and an indicator gene into a host cell; (b) culturing the host cell from step (a); (c) measuring expression of the indicator gene in a target host cell wherein expression of the indicator gene is dependent upon the patient derived segment; and (d) comparing the expression of the indicator gene from step (c) with the expression of the indicator gene measured when steps (a)-(c) are carried out in the absence of the candidate anti-viral drug compound, wherein a test concentration of the candidate anti-viral drug compound is present at steps (a)-(c); at steps (b)-(c); or at step (c)
The expression of the indicator gene in the resistance test vector in the target cell is ultimately dependent upon the action of the patient-derived segment sequences. The indicator gene may be functional or non-functional.
In another aspect this invention is directed to antiretroviral drug susceptibility and resistance tests for HIV/AIDS. Particular resistance test vectors of the invention for use in the HIV/AIDS antiretroviral drug susceptibility and resistance test are identified.
In yet another aspect this invention provides for the identification and assessment of the biological effectiveness of potential therapeutic antiretrovlral compounds for the treatment of HIV and/or AIDS. In another aspect, the invention is directed to a novel resistance test vector comprising a patient-derived segment further comprising one or more mutations on the RT gene and an indicator gene.